Project Summary: The commensal microbiota plays a prominent role in the regulation of immunity. Due to recent advances in our understanding of mechanisms underlying the efficacy of cancer therapies, the gut microbiota has been implicated in modulating the efficacy of various immunotherapies. Tumor cell expression of CD47, a transmembrane protein signaling ?don?t eat me? to phagocytes, promotes immune evasion and tumor relapse. Notably, differential anti-CD47 antibody-induced tumor control has been observed, and our preliminary data indicate that different commensal microbiota produce different anti-tumor responses to CD47 blockade: 1) Tumor-bearing wild-type (WT) mice from Jackson Labs (Jax) responded better to CD47 blockade immunotherapy compared with tumor-bearing mice from Taconic Farms (Tac); 2) antibiotic feeding eliminated the anti-tumor responses in WT mice from Jax; 3) after cohousing with Jax mice, tumor control was enhanced in Tac mice following CD47 blockade; 4) anti-tumor effects were not detected in Germ-Free (GF) WT mice after CD47 blockade; and 5) differences in anti-CD47-induced tumor control were transferred through oral gavage of fecal microbiota. HYPOTHESIS: We hypothesize that the gut microbiota modulate anti-tumor immune responses elicited by CD47 blockade, which could explain the previously observed differential therapeutic responses. Aim 1: Investigate the cellular and molecular mechanisms related to how gut microbiota govern anti- tumor effects induced by anti-CD47 antibody treatment. Aim 2: Identification of microbiota species that mediate antitumor immunity after CD47 blockade. TRANSFORMATIVE POTENTIAL: Checkpoint inhibitors have transformed the treatment of human cancers such as malignant melanoma and lung cancer; however, these nascent immunomodulatory approaches have proven effective thus far in only a subset of patients. While limited preclinical and clinical data suggest that probiotic treatment can promote a gut microbiome that is amenable to the antitumor effects of immunotherapies and chemotherapies, the status and effects of commensal microbiota are currently not widely considered as part of standard practice, even though many cancer patients currently receive pro- and/or antibiotics prior to or during the course of their treatment, for indications such as treatment toxicity and infection. This study has the potential to change clinical practice: determination of the presence or absence of microbiota that potentiates CD47 blockade could improve anti-tumor effects, and expand the patient population that responds to this immune checkpoint inhibitor currently in clinical trials. !